Biomass and nutritional requirements of psychrotrophic bacterial ...

Kieler Meeresforsch., Sonderh. 8, 219-224. Kiel1991

Biomass and nutritional requirements of psychrotrophic bacterial communities in Fram Strait and Western Greenland Sea

Abstract

T. L. Tan and H.-J. Rugar

Alfred-Wegener-Institut fOr Polar- und Meeresforschung Am Handelshafen 12, D-2850 8remerhaven, Germany

During the "Polarstern" expedition ARK Iv/2 in June 1987, water samples from 8 stations were taken to study biomass and substrate utilization of cold adapted bacteria. Bacterial biomasses determined from acridine orange direct counts (AODC) were between 0.4 and 31.4 Ilg CIl, and ATP concentrations amounted from <0.1 to 40 ngll. Colony counts on seawater agar reached only 0.1 % of AODC, but with the MPN-method 1 to 10 % of AODC were recorded. With 14C_ glutamic acid or 14C-glucose as tracer substrate in oligotrophic broth containing 0.5 mg trypticase and 0.05 mg yeast extract per liter of seawater, obligately oligotrophic bacteria could be detected in one water sample. Although incubation was at 2 DC, only psychrotrophic bacteria showing growth temperatures between 1 and 30 DC were obtained. Organic substrate utilizations by 106 isolates were tested at 4 and 20 DC. Most carbohydrates, organic acids, alcohols, and alanine were assimilated at both temperatures, but arginine, aspartate and ornithine were utilized only at 20 DC by almost all strains.

Introduction

For understanding the microbial processes in polar and other cold deep-sea re­gions, a better knowledge of the structure, activity and biomass of bacterial communities in relation to the extreme environmental conditions, like low tem­perature and low nutrient concentrations, is needed.

Oligotrophic bacteria were recorded in the Pacific Ocean and South China Sea (CARLUCCI et al. 1986, ISHIDA et al. 1986), but to our knowledge no investiga­tions exist on the distribution and role of low-temperature adapted oligotrophic bacteria in the arctic ecosystem. The results of our investigations on microbial biomasses, distribution of oligotrophs, and substrate utilizations of the copiotro­phic isolates at low temperatures are presented here.

Material and methods

Water samples were obtained with 20 l-Niskin samplers from 25, 200 and 1000 m depth at 8 stations in Fram Strait and the Western Greenland Sea (Fig. 1).

219

POLARSTERN ARK IV/2

Bacteriological \ ;:" ::;: stations

Fig. 1. Bacteriological stations in Fram Strait and the Western Greenland Sea.

The bacteria were collected on cellulose acetate filters (0.2 iJ.m pore width, 47 mm diameter) and incubated at 2 DC on seawater agar according to ZoBell. Viable counts were estimated after 3 months, and the same plates taken for iso­lation of strains. Substrate utilizations by the isolates were dete r mined as al­ready described (RUGER 1988).

For enumeration of bacteria with the 14C_MPN method (3 flasks per dilution), the copiotrophic and oligotrophic media according to ISHIDA et aJ. (1986) were used; L_[U_ 14C] glutamic acid (specific activity: 148.7 iJ.Ci/mg) or D-[U- 14C] glucose (29.6 iJ.Ci/mg) served as tracer substrate in the oligotrophic medium. Turbidity and liquid scintillation measurements of 14 CO 2 were done after 4 months of incubation at 2 DC.

Bacterial biomasses in 50 and 100 ml of seawater were collected on polycarbon­ate filters (0.1 iJ.m pore width, 47 mm diameter) and the cells preserved with glutaraldehyde vapours in petri dishes. After staining with acridine orange the bacteria were morphologically differentiated into five groups: small coccoid cells, Ilf about 0.28 iJ.fT1, volume 11.5 x 10-' iJ.m'; large coccoid cells, presumably cyanobacteria, Z about 1 iJ.m, volume 523.8 x 10"" iJ.m'; short rods, 0.28 by 0.55 iJ.m, volume 33.9 x 10-' iJ.m; long rods, 0.55 by 2.2 iJ.fT1, volume 522.9 x 10-' iJ.m'; filamentous cells, 0.55 by 5.5 iJ.m, volume 1307.2 x 10-' iJ.m'. Biovolumes were converted into biomasses using the factor 5.6 x 10- 0 gC per iJ.m'. For ATP de­terminations, the microorganisms from 500 ml seawater were filtered, the cells

220

on the filters extracted in 5 ml of boiling Hepes buffer (0.1 M), and the extracts stored at -25 DC (TAN and RUGER 1989).

Results and discussion

Bacterial biomasses from AODC were between 0.4 and 31.4 p.g C/I and ATP con-centrations amounted from < 0.1 to 40 ng/I. Direct counts were between 1.23 and 24.0 x 104 cells/ml. Cyanobacteria were found in almost all water samples; the highest densities were recorded at station no. 227 in 200 m (13.8 x ID') and at station no. 235 (15.1 and 8.1 x ID'), as expressed in the higher biovolumes (Table 1). The AODC results were in the same range as reported for Antarctic Ocean

Table 1. Direct counts, biovolumes and biomasses of bacteria in Fram Strait and the Western Greenland Sea.

Station Depth to Sampling AODC/ml Biovol./ml Biomass/ml ATP/ml No. bottom, m depth, m x ID' x ID' ~m' ng C pg

185 2560 25 9.45 6.92 3.9 10 200 8.92 6.92 3.9 1

1000 3.43 6.80 3.8 < 0.1

195 149 25 10.60 9.18 5.1 2 135 4.50 5.64 3.2 0.3

223 2629 25 2.27 3.88 2.2 29 200 1.33 0.75 0.4 0.4

227 747 25 1.23 3.0 10 200 11.30 17.8 0.5

235 403 25 24.00 31.4 7 200 19.70 22.1 0.2

242 2466 25 9.37 12.17 6.8 40 200 5.91 11.62 6.5 1

1000 1.97 2.31 1.3 < 0.1

248 1450 25 13.14 18.56 10.4 18 200 1.92 1.58 0.9 1

1000 9.68 19.88 11.1 < 0.1

253 315 25 4.52 8.96 5.0 5 200 5.10 3.76 2.1 8

AODC: acridine orange direct counts; ATP: adenosine triphosphate

water samples by SIMIDU et al. (1986). The cyanobacteria in the East Greenland Current came presumably from the warmer Atlantic Intermediate Water mixed

221

with Polar Water (GRADlNGER and LENZ 1989). ATP concentrations in Antarc­tic waters around Elephant Island and west of the Antarctic Peninsula in Austral winter (VOSJAN et al. 1987) were somewhat higher than found in Fram Strait and Western Greenland Sea in summer time.

Table 2. Densities of cultivable copiotrophic and oligotrophic bacteria in Fram Strait and the Western Greenland Sea.

Station Sampling CFU/ml MPN/ml MPN/ml MPN/ml No. depth, m copiotrophs copiotrophs oligotrophs, oligotrophs,

facultative obligate

185 25 2.50 x ID' 348 x ID' 9.1 x ID' 0 200 1.60 X ID' 33 x ID' 23.0 x ID' 0

1000 2.30 X ID' 17 x ID' 15.0 x ID' 0

195 25 0.35 x 102 46 X ID' 9.1 x 10 2 0 135 0.14 x 10 5 X ID' 0 0

223 25 0.48 x ID' 348 x 10 2 3.6 X ID' 200 0.47 x ID' 5 x 10 2 3.6 X ID'

1000 0.71 x 102 n.d. n.d. n.d.

227 25 0.93 x ID' 49 x ID' 0 0 200 0.32 X ID' 7 x ID' 9.1 x ID' 0

235 25 0.93 x ID' 84 x ID' 3.6 x ID' 0 200 1.60 X ID' 23 x ID' 15.0 x ID' 0

242 25 1.10 x ID' 17 x ID' 3.6 x 10 2 0 200 1.90 X 102 8 X ID' 7.3 x 10 2 0

1000 1.50 X 102 n.d. n.d. n.d.

248 25 1.10 x ID' 542 x ID' 3.6 x 10' 0 200 1.10 X ID' 8 x ID' 9.1 x ID' 0

1000 6.20 x 1 13 X ID' n.d. n.d.

253 25 0.46 x ID' 70 x 10 2 7.2 X ID' 0 200 0.33 X ID' 70 x 10 2 n.d. n.d.

CFU: colony forming units; MPN: most probable number; n.d.: not determined

Colony counts on seawater agar were between 0.14 and 6.20 x 10'/ml, but with the MPN-method cell densities were 1-2 orders of magnitude higher (Table 2). This relation between the two methods is in agreement with SIMIDU et al. (1986). Colony counts from the Gulf of Alaska were lower than those reported here, but the plates were already evaluated after 3 weeks incubation at 5° C (HAUXHURST et al. 1980). In contrast to ISHIDA et al. (1986), who reported obligately oligotrophic bacteria to be the dominant populations in the South China Sea and West Pacific Ocean, obligately oligotrophic bacteria were found only in one water sample from the Western Greenland Sea.

222

UI Z :;;: a:

'::1 ~ UI

0 UJ

!;i ...J 0 !!! IL 0 ~ 100 z UJ U a: UJ Q.

50

13 STRAINS

R ... -. ..

9 STRAINS

GlU GAL CELL SAC ACET sue GlY MAN ALA ARG ASP ORN

UTILIZATION OF ORGANIC SUBSTRATES ~ ___ ~ ... _

DEPTH: 25m

DEPTH: 200m

DEPTH: 1000m

Although incubation was at 2 DC, only psychr.otrophic bacteria showing growth temperatures between 1 and 30 DC could be isolated (s. BAROSS and MORIT A 1978). Organic substrate utilizations by 106 strains were tested at 4 and 20 DC. Most carbohydrates, organic acids, alcohols, and alanine were assimilated at both temperatures, but arginine, aspartate and ornithine were used only at 20 DC by almost all strains. n example for substrate utilizations by the bacterial com­munities from one station IS shown in F"ig. ~ Similarly, most of the bacteria from the Gulf of Alaska (HAUXHURST et al. 1980) were psychrotrophs and 20 representative strains utilized organic substrates at both 5 and 20 DC. On the contrary, isolates from deep-sea sediments of the western tropical Atlantic uti­lized most organic substrates only at 4 DC, but not at 20 DC (RUGER 1988). NORKRANS and STEHN (1978) also reported that a great proportion of their sediment bacteria from the Norwegian and Western Greenland Sea were psychro­philic organisms showing maximum growth temperatures of 20 DC or below.

Acknowledgements

We thank Dr. K.-G. Barthel, Institut fUr Meereskunde Kiel, for valuable discus­sions and helping to get the water samples, Annegret Mi:idler, Karin Springer and Christa Summa for their expert technical assistence and Marion PuIs for prepar­ing the graphs. This work was supported in part by the Deutsche Forschungsge­meinschaft (Ta 63/7).

223

Contribution No. 358 of Alfred-Wegener-Institut fUr Polar- und Meeresforschung, Bremerhaven.

References

BAROSS, J.A. and R.Y. MORITA, 1978. Microbial life at low temperatures: Ecological aspects. In: D.J. KUSHNER: Microbial life in extreme environ­ments. Academic Press London. 9-71-

CARLUCCI, A.F., S.L. SHIMP and D.B. CRAVEN, 1986. Growth characteristics of low-nutrient bacteria from the north-east and central Pacific Ocean. FEMS Microbiol. Ecol. 38, 1-10.

GRADINGER, R. and J. LENZ, 1989. Picocyanobacteria in the high Arctic. Mar. Ecol. Prog. Ser. 52, 99-101-

HAUXHURST, J.D., M.I. KRICHEVSKY and R.M. ATLAS, 1980. Numerical tax­onomy of bacteria from the Gulf of Alaska. J. Gen. Microbiol. 120, 131-148.

ISHIDA, Y., M. EGUCHI and H. KADOT A, 1986. Existence of obligately oligotro­phic bacteria as a dominant population in the South China Sea and the West Pacific Ocean. Mar. Ecol. Prog. Ser. 30, 197-203.

NORKRANS, B. and B.O. STEHN, 1978. Sediment bacteria in the deep Norwe­gian Sea. Mar. Bioi. 47, 201-209.

RUGER, H.-J., 1988. Substrate-dependent cold adaptations in some deep-sea sediment bacteria. System. Appl. Microbiol. 11, 90-93.

SIMIDU, U., K. KOGURE, K. FUKAMI and C. IMADA, 1986. Heterotrophic bac­terial flora of the Antarctic Ocean. Mem. Nat!. Inst. Polar Res, Spec. Issue 40, 405-412.

TAN, T.L. and H.-J. RUGER, 1989. Benthic studies of the Northwest African upwelling region: bacteria standing stock and ETS-activity, ATP-biomass and Adenylate Energy Charge. Mar. Ecol. Prog. Ser. 51, 167-176.

VOSJAN, J.H., G. NIEUWLAND, W. ERNST and T. BLUSZCZ, 1987. Shipboard comparison of two methods of extraction and measurements of ATP applied to Antarctic water samples. Neth. J. Sea Res. 21, 107-112.

224